Flowers and Leaves Extracts of Stachys palustris L. ExhibitStronger Anti-Proliferative, Antioxidant, Anti-Diabetic, andAnti-Obesity Potencies than Stems and Roots Due to MorePhenolic Compounds as Revealed byUPLC-PDA-ESI-TQD-MS/MSSabina Lachowicz-Wisniewska 1,2,3,* , Anubhav Pratap-Singh 3 , Ireneusz Kapusta 4 , Angelika Kruszynska 5,Andrzej Rapak 5 , Ireneusz Ochmian 2 , Tomasz Cebulak 4 , Wioletta Zukiewicz-Sobczak 1
and Paweł Rubinski 1
1 Department of Food and Nutrition, Calisia University, 4 Nowy Swiat Street, 62-800 Kalisz, Poland;[email protected] (W.Z.-S.); [email protected] (P.R.)
2 Department of Horticulture, West Pomeranian University of Technology in Szczecin, 71-434 Szczecin, Poland;[email protected]
3 Faculty of Land and Food Systems (LFS), The University of British Columbia, Vancouver Campus 213-2205East Mall, Vancouver, BC V6T 1Z4, Canada; [email protected]
4 Department of Food Technology and Human Nutrition, College of Natural Science, Rzeszow University,4 Zelwerowicza Street, 35-601 Rzeszow, Poland; [email protected] (I.K.); [email protected] (T.C.)
5 Laboratory of Tumor Molecular Immunobiology, Ludwik Hirszfeld Institute of Immunology andExperimental Therapy, Polish Academy of Sciences, 53-114 Wroclaw, Poland;[email protected] (A.K.); [email protected] (A.R.)
Abstract: The present work aims to assess the biological potential of polyphenolic compounds indifferent parts (flowers, leaves, stems, and roots) of Stachys palustris L. Towards secondary metabolitesprofile, 89 polyphenolic compounds (PCs) were identified by UPLC-PDA-ESI-TQD-MS/MS, witha total average content of 6089 mg/100 g of dry matter (d.m.). In terms of biological activity,antioxidant activity (radical activity, reducing power), digestive enzyme inhibitory (α-glucosidase,α-amylase, pancreatic lipase) effect, and antiproliferative activity (inhibition of cell viability andinduction of apoptosis in different human cancer cell lines) were explored. Leaves, flowers, stems,and roots of S. palustris L. have not been studied in this regard until now. Vescalagin and cocciferind2, isoverbascoside (verbascoside), luteolin 6-C-glucoside, luteolin 6-C-galactoside, apigenin 6-C-glucoside, (−)-epicatechin, ellagic acid, and malvidin 3-O-diglucoside were detected as mainingredients in the studied parts. Methanolic extract of S. palustris L. leaves and flowers revealedthe highest amount of PCs with the strongest antiradical (18.5 and 15.6 mmol Trolox equivalent(TE)/g d.m., respectively) and reducing power effects (7.3 and 5.6 mmol TE/g d.m.). Leaf extractsexhibited better α-amylase and pancreatic lipase inhibition effects, while flower extracts exhibitedbetter α-glucosidase inhibition effect. Regarding antiproliferative activity, extracts of the leaves andflowers significantly reduced cell viability and induced a high level of apoptosis in human lung,pancreatic, bladder, and colon cancer cell lines, as well as in human acute myeloid leukemia; whereasthe extracts from stems and roots revealed the weaker effects. The results of this work showedanti-proliferative and potentially anti-diabetic, anti-obesity properties of S. palustris L., especially forflowers and leaves, which may have wide potential applications in the functional food, special food,pharmaceutical, cosmetics industries, and/or in medicine.
Keywords: bioactive compounds; in vitro biological potency; medical plant; marsh woundwort
In recent years, edible plants, forgotten plants, wild plants, as well as medical plantshave emerged as the potential sources of secondary metabolites for therapeutic interven-tions [1], which has opened doors for their use as active ingredients in food, pharmaceutical,cosmetics, and/or medical products.
Stachys palustris L. (sect. Stachys; family Lamiaceae, subfamily Lamioideae), otherwiseknown as marsh woundwort, is one such edible plant found in both lowlands and moun-tains, which has been used in traditional medicines, such as emetic, antiseptic, nervine,sedative, antispasmodic, emmenagogue, hemostatic, vulnerable, expectorant, and tonicagent [2–5]. However, it is most effective in treating internal and external hemorrhages,cramps, and joint pains, as well as the treatment of gout [2]. This plant can grow up to 1 mtall, has square stems, purplish-red flowers arranged in whorls, opposite leaves, and dryfour-chambered schizocarp fruit [2].
The biosynthesis of polyphenolic compounds is an intensive process, largely condi-tional on abundant factors linked with the plant and their environment. The distributionand concentration of bioactive compounds in different underground and above-groundparts of the plant can be strongly varied. This is mainly tied to the role of polyphenols inthe growth phase and in plants’ life cycle [6]. Previous research has shown that S. palustrisL. plants exhibit a high total polyphenol content of about 213 mg gallic acid equivalent(GAE)/g dry matter (d.m.), high antiradical activity against DPPH radical, and promisinganti-proliferative properties against cervix adenocarcinoma cells (HeLa) [2,7]. On the otherhand, the chromatographic analysis showed the presence of only eight compounds belong-ing to the group of phenylethanoid glycosides, isoscutellarein derivatives, phenolic acid,and iridoids [2].
Despite the above data and traditional knowledge about S. palustris L. being a highnutraceutical potential, little is known about the secondary metabolites profile and biologi-cal activity of edible leaves, flowers, stems, and roots of S. palustris L. [6,7]. Therefore, thisstudy aimed to assess the profile and amount of polyphenolic compounds by UPLC-PDA-ESI-TQD-MS/MS and their in vitro biological activity (antioxidant and anti-proliferativeactivity). Results, on the polyphenol profile and anti-proliferative activity, as well asinhibitory activity against digestive enzymes, could be interesting for potential applica-tions in functional foods, natural health products, pharmaceuticals, cosmetics industries,and/or medicine.
2. Results and Discussion2.1. Profile of Polyphenolic Compounds
The UPLC–PDA-ESI–TQD–MS/MS fingerprint of Stachys palustris L. flowers, leaves,stems and roots (Table 1, and Figure S1) discovered the presence of 89 polyphenolic in-gredients based on their UV maxima, m/z, [M-H]− and [M-H]+, retention times, peakareas, available data, and standards. Amongst identified compounds, 40 hydrolysabletannins, 16 phenylethanoid glycosides (PhGs), 4 anthocyanins, 1 flavan-3-ol, 6 phenolicacids, 1 flavonols, and 21 flavones were found. However, the qualitative composition ofthe tested bioactive ingredients was strongly dependent on the extracted part of the plant;with 56, 55, 50, and 49 compounds identified, respectively, in flowers, roots, leaves, andstems of S. palustris L (Tables 1 and 2). It is worth emphasizing that thus far only eightcompounds had been identified in S. palustris L. belonging to secondary metabolites, suchas verbascoside, echinacoside, two isoscutellarein derivatives, monomelittoside, chloro-genic acid, harpagide, and its derivative 8-O-acetyl-harpagide [2]. However, in our study,compounds, such as monomelittoside, chlorogenic acid, harpagide, and its derivative8-O-acetyl-harpagide [2], were not observed. Thus, apart from the compounds confirmedby Venditti et al. [2], the remaining compounds were detected in the flowers, leaves, stems,and roots extracts of S. palustris L. for the first time.
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Table 1. Characterization of polyphenolic compounds in Stachys palustris L. by LC–PDA-ESI–TQD–MS/MS.
Degree of polymerization (DP) 2.96c 2.07d 3.72b 4.74a
The total sum of phenolic compounds 8544.63b 9252.11a 4938.76c 1622.94da Values that are expressed as the mean (n = 3) ± standard deviation and different letters (between morphologicalparts) within the same row indicate statistically significant differences by Duncan’s test (p < 0.05); b nd, notidentified.
2.1.1. Hydrolysable Tannins
The main class of polyphenolic compounds identified in the flowers, leaves, stems,and roots of S. palustris L. was hydrolysable tannins. In this study, S. palustris L. was foundto contain 40 compounds of this class, including 36 hydrolysable tannins in the flowers,31 in the leaves, 32 in the stems, and 22 in the roots. This group contains the derivativesof gallic acid, ellagitannins, and gallotannins [8]. Gallic acid is known to dimerize intoellagic acid formed by intramolecular dilactonization of HHDP acid (hexahydroxydiphe-noyl acid). Ellagitanins, constituting the most abundant fraction of hydrolysable tannins,are formed by the oxidative coupling of adjacent galloyl fractions from gallotannins [8,10].The group of HHDP is further divided into two groups: a NHTP (nonahydroxytriph-enoyl) group by coupling to the next galloyl group, or a Chebuloyl group by doubleoxidation [8,10]. The structure of thirty-four compounds were confirmed, based on iden-tification of their fragmentation pattern on the basis of fragmentation earlier describedin the Chestnut of Castanea sativa by Miller [8], the different plant parts of Terminalia ar-juna [9], the fraction of Sanguisorba officinalis L. [11], the different parts of myrtle berry fromItaly [12], Fragaria vesca L. berries [13], Terminalia chebula fruits [14], plant medical [15,16],Myrtaceae family [17], Genista tinctoria L. [18], and the leaves of Phyllagathis rotundifolia [19].These compounds were identified as grandinin, two grandinin derivatives, roburin E(m/z 1065), fourteen castalagin/vescalagin isomers (m/z 933), four pedunculagin isomer[diHHDP-glucose] (m/z 783), two casuarinin [diHHDP-galloyl-glucose] (m/z 935), casuar-ictin [galloyl-DiHHDP-glucose] (m/z 935), two chebulanin (m/z 651), geraniin isomer(m/z 951), pentagalloyl-glucose (m/z 939), two tellimagrandin I [digalloyl-HHDP-glucose](m/z 785), and four trigalloyl-HHDP-glucose (m/z 937).
The fragmentation of the above hydrolysable tannins resulted in losses of typicalresidues such as gallic acid ([M-H-170]−), galloyl ([M-H-152]−), HHDP ([M-H-302]−),HDDP glucose ([M-H-482]−), galloyl-HDDP-glucose ([M-H-634]−), or galloyl-glucose ([M-H-332]−) residues [8,10,11]. According to Esposito et al. [8], the compound of chebulagicacid (galloylo-chebuloyl-HHDP-glucose) contains chebuloyl group generated by the oxi-dation of HHDP residues and further supported by the loss of carboxylic and chebuloylgroups. In addition, the compound cocciferin d2 isomer (HHDP-NHTP-glucose-galloyl-diHHDP-glucose), which is a dimer ellagitannin, was noted as [M-2H]2− (double chargedpseudomolecular ion). Sanguiin H-10 isomers [M-H]− at m/z 1567 were identified on thebasis of data published by Kool et al. [20] in Boysenberry (Rubus loganbaccus × baileyanusBritt.), and by Mullen et al. [21] during the fragmentation MS/MS loss of two HHDP (302mass unit (m.u.), glucosyl (162 m.u.), and galloyl (170 m.u.) moieties.
2.1.2. Flavanones
The compounds belonging the group of flavones usually subsist as glycosides andseldom as free aglycones [37]. In the full-scan LC-MS/MS, the deprotonated pseudomolec-ular ions [M-H]− of luteolin, apigenin, isoscutellarein, and chrysoeriol (m/z 285.0, m/z269.0, m/z 285.0, and m/z 299, respectively) were observed [33]. A total of 21 compoundsare reported in different parts of S. plasturis L., including 7 flavones in the flowers, 9 in theleaves, 8 in the stems where apigenins and luteolins dominated, and 15 in the roots whereisoscutellarein and chrysoeriol dominated. The pathway for flavones C-glycosides showed
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the decomposition of glycan with a loss of neutral residues with the decomposition of aflavonoid moiety associated to the residual part of glycan [38]. These compounds for thegenus Stachys are considered important chemosystematics markers [25].
The first class of flavones was luteolin ([M-H]− at m/z 447) determined based oncharacteristic UV absorption maxima at 269 and 349 nm. According to Lin et al. [37], thesecompounds are characterized by two UV spectral maximums: 250–300 nm (band I), and342–350 nm (band II). Based on the fragmentation of the peak that indicated the presence ofa C-glycosylated derivative, these compounds were confirmed as luteolin 6-O-galactosideand luteolin 6-O-glucoside (previously identified in the Elaeis guineensis Jacq.) [33,34].
Apart from luteolins, four apigenin compounds (m/z 431, 577, and 635) were also iden-tified based on the UV spectrum at 260 and 336 nm. Two compounds with molecular ion atm/z 431 and the fragmentation of MS/MS indicative of a C-glycosylated derivative and lossof glucosyl (162 m.u.) moiety were identified as apigenin 6-C-galactoside and apigenin 6-C-glucoside, previously noted in Cyclanthera pedata leaves, fruits, and dietary supplements [35].The compound with pseudomolecular ion at m/z 635 was tentatively identified on thebasis of fragmentation earlier described in Stachys parviflora L. [26] as apigenin acetylallosyl-glucoside. Mass spectra of apigenin with p-coumaric acid derivative showed molecularion at m/z 577 and was confirmed based on previous results for Stachys byzantina [36] andStachys bombycina [31] as apigenin 7-O-β-D-(6-p-coumaroyl)-glucopyranoside.
Two compounds belonging to chrysoeriol derivatives with pseudomolecular ion atm/z 667 and m/z 677 were confirmed based on the fragmentation pathway described inthe Stachys subgenus [32] and Stachys bombycina [31] and were identified as chrysoeriol7-O-acetylallosylglucoside and chrysoeriol-acetyl-allopyranosyl-glucopyranoside.
The last class of flavones were twelve isoscutellarein derivatives with the pseudo-molecular ion [M-H]− m/z 667, m/z 707, m/z 665, and m/z 651. The presence of Aglycon[A-H]− fragment and D-allose, which is characteristic of a large group of Stachys plants,was noted [25]. As constituents acetylated on the internal or external allose unit showingthe intermediate ions [M-180]− or [M-180-OAc]− [25], these compounds were identifiedas isoscutellarein-acetylallosyl-(glucopyranoside)apiose isomers (nine compounds), 4′-O-methylisoscutellarein-acetyl-allosyl-glucopyranoside, and their isomers similar to Stachysrecta L. [25] and Stachys parviflora L. [26].
2.1.3. Phenylethanoid Glycosides (PhGs)
Phenylethanoid glycosides (PhGs) are appreciated for a strong biological potency [39,40].The typical spectra exhibited UV-Vis maxima between 320–340 nm [39]. 16 PhG com-pounds, including 2 in the flowers, 3 in the leaves, 2 in the stems, and 13 in the roots,were detected in S. palustris L. The detected PhGs showed deprotonated molecular ions[M-H]− at m/z 785, m/z 755, m/z 623, m/z 799, m/z 769, m/z 651, m/z 755, and m/z669. During the fragmentation of the above, PhGs showed losses of typical residues, suchas the neutral loss of the caffeoyl residue (162 m.u.), p-coumaric moiety (176 m.u.), therhamnose residue (146 m.u.), the glucose residue (162 m.u.), the COCH2 group (42 m.u.), aCH2 radical (14 m.u.), and the methoxy group (30 m.u.) [25,26,39,40].
Compounds with pseudomolecular ions at m/z 785, m/z 623, m/z 755, and m/z669 were confirmed as betonyoside E, two B-OH-Forsythoside B methylether isomers,isoacteoside, two forsythoside B, and their isomer based on stachysoside E, previouslyisolated from Stachys recta L. [25], Stachys parviflora L. [26], Stachys officinalis L. [27], andStachys alopecuros L. [2].
Mass spectra for two compounds were tentatively assigned as echinacoside (m/z 799)and cistanoside A (m/z 785), previously described in Cistanche deserticola [22] and Cistanchearmena [23].
Three compounds with pseudomolecular ions at m/z 756 and 637 was tentativelydetected as two stachysoside A and leucoseptoside A, with a characteristic fragmentationpathway presented in Lagopsis supina [28].
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The last three compounds belonging to PhGs were tentatively specified on the ba-sis [24] noted in Phlomis species. Two compounds having pseudomolecular ions at m/z 769,and on the basis of fragmentation MS/MS the losses of p-coumaric residue, pentose, andglucose moiety, were tentatively assigned as alyssonoside and alyssonoside isomer [24].Compounds, such as samioside (m/z 755) and martynoside (m/z 651), were assigned onthe losses of glucose, pentose, rhamnose moiety, and p-coumaric and rhamnose moiety,according to the fragmentation pattern determined by Kirmizibekmes et al. [24].
2.1.4. Other Phenolic Acids, Anthocyanins, Flavonol, and Flavan-3-ol
The last groups of polyphenolic compounds identified in the different parts of S.palustris L. were phenolic acids, anthocyanins, flavonol, and flavan-3-ol. Among detectedphenolic acids, three compounds presented similar maximum absorbance at 325 nm, whichis typical for derivatives of dicaffeoylquinic acid [25]. All of them had common pseudo-molecular ions at m/z 515. The first compound was detected as 3,5-di-affeoylquinic acidcompared with the standard. The next two compounds noted the characteristic ions at m/z173, which indicate the presence of quinic acid in position four. Thus, these compoundswere determined as 3,4-di-caffeoylquinic and 4,5-di-caffeoylquinic acids, respectively, onthe basis of the order of elution [25]. The next three phenolic acid tentatively assignedas ellagic acid (m/z 301), ellagic acid glucoside ([M-H-463]−), and ellagic acid pentoside([M-H-461]−) were previously determined in Stachys officinalis [33].
All anthocyanins and flavonol assigned in S. palustris L. was noted in just flowers forthe first time. In the full-scan LC-MS/MS, the deprotonated molecular ions [M-H]− ofdelphinidin, malvidin, and cyanidin (m/z 303, m/z 331, and m/z 287, respectively) [29]were detected. These compounds were detected as delphinidin 3-O-glucoside, malvidin3-O-diglucoside, cyanidin 3-O-glucoside, and malvidin 3-O-acetylglucoside on the basis oftheir fragmentation pathways [29]. One flavonol (deprotoned molecular ion at m/z 285)was specified as kaempferol hexose glucuronide from the losses of hexose moiety (162 m.u.)and glucuronide moiety (176 m.u.) [30]. The existence of (−)-epicatechin was confirmedvia the standard.
2.2. Content of Polyphenolic Compounds and Polymeric Procyanidins
Polyphenols are a very important group of secondary metabolites because they exhibita wide range of health benefits and biological activities [41]. A statistical test on one-factor analysis indicated a significant effect (p < 0.05) of research plant parts on the totalpolyphenol content (TPC). The average TPC in the S. palustris L. was 6090 mg/100 gd.m. (Table 2). The highest amount of secondary metabolites were measured in theleaves (9252 mg/100 g d.m.), and this value was 1.9 and 5.7 times higher than in the stemsand roots.
The main group of the analyzed polyphenols detected in the flowers, leaves, stems,and roots of S. palustris were hydrolysable tannins (constituting an average of 82.9% of allpolyphenolic compounds) > flavones (8%) > flavan-3-ols (monomers and polymers; 5%) >phenylethanoid glycosides (3.3%) > phenolic acids, anthocyanins, and flavanols (<0.8%).Compared with other Stachys species, hydrolysable tannins were also a predominantgroup detected in S. cretica ssp. anatolica [42]. Nonetheless, higher TPC detected in theflowers and leaves is because these organs actively metabolize these compounds duringphotosynthesis [43,44].
A similar trend for TPC was noted in the studies on different parts of Astragalusmacrocephalus subsp. finitimus [45]. In the S. palustris L. isolated from Hungary and France,the TPC was 17,630 and 24,980 mg GAE/100 g d.m., respectively [2], and was 1.8 and2.5 times higher than in the leaves of S. palustris L. from Poland. In turn, the TPC of ourextracts were significantly higher than those reported for other Stachys species (rangingfrom 430 mg GAE/100 g d.m. in S. trinervis to 4450 mg GAE/100 g d.m. in S. fruticulosa)from Iran by Khanavi et al. [46]. According to Oracz et al. [47], TPC may be influenced bymany factors, e.g., origin, soil, weather conditions, analytical method, and the preparation
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of test samples. Carev et al. [42] reported that methanol extracts of S. cretica ssp. anatolicafrom Turkey showed 4330 mg/100 g d.m. TPC assessed by LC–ESI–MS/MS and 1.6 timeslower TPC by spectrophotometric method [42]; the results were significantly lower thanthose reported in the flowers and leaves noted in our work. Likewise, during the analysis ofS. cretica ssp. Vacillans, the record showed 3604 mg/100 g d.m. TPC using the HPLC system,and this value was 2.3 times lower compared to the spectrophotometric method [41]. Onthe other hand, the total concentration of TPC isolated from stems of S. officinalis L. were6120 mg GAE/100 g dry extract [33], 1610–3330 mg GAE/100 g [48], which were 1.2 timeshigher and 2 times lower compared to stems of S. palustris L. The total amount of bioactivecompounds extracted from leaves and roots of Stachys officinalis L. from Czech Republicwere, respectively, 7495–8050 mg GAE/100 g d.m. and 2286–3164 mg caffeic acid/100 gd.m. (depending on the cultivar tested) [49]. TPC in the S. recta was 2050 mg/100 g d.m.and was around 4.8 times lower than in the leaves of S. palustris L. [25].
The most abundant group of tested parts of S. palustris L. contained a range of 1083 to7945 mg/100 g d.m. for roots and flowers, respectively. The total tannins content identifiedin the different organs of Calluna vulgaris L. Hull in flowering time were 5, 4.3, and 3 timeslower than our data for flowers, leaves, and stems [43]. In addition, the predominantcompounds among 40 hydrolysable tannins were vescalagin and cocciferin d2 constituting21% and 19%, respectively, in the leaves and 37% and 21% in the roots. This was alsoconfirmed in the study by Esposito et al. [8] and Singh et al. [9].
The concentration of the second numerous group [42] ranged from 138.9 in the flowersto 1406.0 mg/100 g d.m. in the leaves. The most abundant compound in the flowerswere luteolin 6-C-galactoside and 4′-O-methylisoscutellarein-glucoside-rhamnoside (con-stituting 33 and 26% of all flavones); in the leaves it was luteolin 6-C-galactoside andapigenin 6-C-glucoside (constituting 33 and 28%); in the stems it was luteolin 6-C-glucosideand apigenin 6-C-glucoside (constituting 33 and 27%); in the roots it was isoscutellarein-acetylallosyl-(glucopyranoside)apiose (constituting 55%). In turn, in studies by Carevet al. [42] the major compound in S. cretica ssp. anatolica was apigenin-7-glucoside and thiswas 2540 mg/100 g d.m. The total of flavonoids as quercetin equivalent in S. tmolea [50]and in S. cretica ssp. vacillans [41] were 500 and 5010 mg/100 g; in Stachys cretica subsp.kutahyensis there was 4020 mg/100 g extract [51]. In addition, apigenin and luteolin were re-ported to reveal a high anxiolytic potency in rats [52] and have high anticancer, antioxidant,and anti-inflammatory activities [50,53].
The next quantitatively important group was flavan-3-ols, including (−)-epicatechinas a monomer (constituting 5% of all flavan-3-ols) and polymeric procyanidins (PP; consti-tuting 95%). The average amount of (−)-epicatechin in measured parts of the plant was12.6 mg/100 g d.m. and PP—254.3 mg/100 g d.m. The (−)-epicatechin of measured partsmay be organized in the following sequence: flowers > stems > leaves, and for PP: leaves >flowers > roots > stems. A similar trend for PP was noted in the different tested organs ofRumex crispus L. and Rumex obtusifolius L. [6]. The total of catechins as catechin equivalentin Stachys marrubiifolia viv. the leaf was 40 mg/100g extract [54]. The lowest amount offlavan-3-ols was detected in the stems and also confirmed by Feduraev et al. [6], probablyby the inside metabolic action of the tissues and cells and the molecular constitution of theexudate carried by the phloem channels [6]. In addition, the alkaline solution of the centralcavity of stems is exposed to oxidation PCs, including flavan-3-ols [6].
Moreover, the most opulent in PhGs were the roots, and the content was 209.5 mg/100 g d.m.,and this amount was an average of 34 times higher than in the rest parts of S. palustris L.The main compound detected in the roots was isoverbascoside (verbascoside). This wasalso confirmed in the research on S. cretica ssp. anatolica by Carev et al. [47], on S. creticasubsp. mersinaea by Bahadori et al. [41], on S. tmolea by Elfalleh et al. [50], and on Stachyscretica subsp. kutahyensis [51]. During the measurement of S. recta, the content of PhGs was607 mg/100 g d.m. and the concentration was around three times higher than in the stemsof S. palustris L. [25]. In addition, the biological activity of verbascoside was confirmed,
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such as, for example, anti-tumor, anti-inflammatory, anti-radical, and anti-thromboticeffects [50].
In turn, the phenolic acids were the most abundant in the flowers compared to the restparts of S. palustris L. tested, and their amount was 2.2, 3, and 8 times higher comparedto the rest fractions. The total content of phenolic acids measured in the S. recta was709 mg/100 g d.m. [25] and was significantly higher than our results. The anthocyanins,and flavonols were noted in the flowers. These compounds present less than 1% of allPCs in the measured extract of S. palustris L. The total of anthocyanins and flavonols asquercetin and cyanidin 3-glucoside equivalent in the Stachys marrubiifolia viv. leaves were537 and 70 mg/100 g extract [54]. According to Bahadori et al. [41], the phenolic acidcontent measured in S. cretica subsp. mersinaea was 401 mg/100 g extract; in S. tmolea was118 mg/100 g [50]. In turn, anthocyanin compounds, in addition to the health benefits, areresponsible for the color of the raw material. According to the analysis, the flowers are thedarkest and red with the addition of dark yellow color (Figure S1). In addition, the NAIindex indicates an intense dark red pigment located in the flowers. On the other hand, thetype of anthocyanins identified indicates a reddish-purple color. Of course, depending onthe pH, it can change [2,29]. This is consistent with the botanical color of the flowers of theplant under study [55]. The root also seems interesting because it showed a dark red colorwith the addition of dark yellow [55].
2.3. In Vitro Biological Activity2.3.1. Antiradical and Reducing Potential
The antioxidant properties of S. palustris L. was assessed as reducing power (FRAPassay) and radical scavenging activity (ABTS assay). Results indicate that significantdifferences were noted between parts of the research plant. Table 3 suggests that theflowers and leaves had the highest radical scavenging activities (18.5 and 15.6 mmol TE/gd.m. respectively) and the highest FRAP reduction potential (5.6 and 7.3 mmol TE/g d.m.,respectively). The ability to scavenge synthetic ABTS radicals determined for the roots andstems was about 2 and 3.8 times and 4.4 and 4.5 times weaker compared to the flowers andthe leaves. Similar conclusions were found for the iron (III) reduction capacity, which was5.3 and 18.5 times and 4 and 14.3 times weaker compared to the flowers and the leaves.
a Values that are expressed as the mean (n = 3) ± standard deviation and different letters (between morphologicalparts) within the same row indicate statistically significant differences (p < 0.05).
The results indicate that the flowers and the leaves are more effective at eliminatingexcess reactive oxygen species (ROS) that cause oxidative stress in the body compared to theroots and the stems [41,45]. When assessing the reducing power in S. anisochila, S. beckeana,S. plumosa, and S. alpina spp. Dinarica, a value of 1.9, 1.8, 0.5, and 1.4 mmol Fe2+/g d.m.has been reported [56]. Likewise, significantly lower values of antioxidant activity for theFRAP test were noted for the areal part of S. trinervis, S. byzantina, S. setifera, S. subaphylla, S.turcomanica, S. inflata, S. laxa, S. persica, and S. fruticulosa [45]. In S. tmolea (the areal part), theantiradical activity and reducing power was 32.3 and 41.9 mg TE/g d.m., respectively [50].In studies conducted by Benabderrahim et al. [51], the antioxidant activity measured byABTS and FRAP assays in S. cretica subsp. Kutahyensis were 175.8 and 239.1 mg TE/g ofextract. The results obtained by Bahadori et al. [41] in Stachys cretica subsp. Mersinaea were292.7 and 236.4 mg TE/g of extract for ABTS and FRAP tests, respectively. Whereas, in
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the extracts of S. cretica ssp. Anatolica for antiradical activity and reducing power were112.2 and 127.2 mg TE/g of extract [42]. Moreover, our obtained results were not directlycomparable. Besides, the research by Saravanakumar et al. [57] and Yadav et al. [58] showsthat the differences in the value of antioxidant activity may significantly depend on thepurity of solvents and their polarity, extraction procedure, and fractionation methods.
2.3.2. In Vitro Enzyme Inhibition
Digestive enzymes, such as pancreatic lipase, are involved in fat metabolism and sup-port the digestion of dietary fats into fatty acids. A-glucosidase and α-amylase are engagedin the carbohydrate metabolism and break down complex sugars into monosaccharidesand oligosaccharides [57,59]. We analyze different parts of the plant with the digestiveenzyme inhibitory properties to evaluate potential anti-obesity and anti-diabetic properties(Table 3).
The higher pancreatic lipase enzyme inhibitory activity was reported for leaves ofS. palustris L. In addition, the flowers and leaves noted the higher α-amylase and α-glucosidase enzyme inhibitory properties. The least ability to inhibit the activity of digestiveenzymes was noted for the roots; the ability to inhibit the activity of α-amylase was onaverage 3.5 times weaker compared to the leaves and the flowers, while the α-glucosidaseactivity was 3 times weaker. The ability to inhibit pancreatic lipase activity was noted forboth the roots and the flowers.
The higher enzyme (α-amylase and α-glucosidase) inhibitory activity was evaluatedin Stachys japonica of methanol extract, and the result was 7.43 ug extract/ug acarboseequivalent (ACE) [57]. Whereas the methanol extract from S. cretica subsp. Smyrnaea [41,60],S. cretica subsp. Mersinaea [41,60], and S. cretica subsp. Kutahyensis [51] had α-amylaseinhibitory activity at the level 61.5, 418.6, and 315.5 mg ACE/g extract, respectively [50].Furthermore, the inhibitory activity against α-glucosidase for Stachys cretica subsp. Mersi-naea was 734.5 mg ACE/g of methanol extract [41]. Another study indicated that theα-amylase inhibition activity for S. iberica subsp. Iberica var. densipilosa and S. byzantina was219.5 and 200.1 mg ACE/g, respectively [61].
We conclude that the leaves and flowers of S. palustris L. have PP and PA compoundsthat show potentially high antidiabetic activities and may be used for the inhibition ofthese enzyme activities. To our knowledge, the inhibition of α-amylase and α-glucosidaseactivity had not been reported before for S. palustris L. extracts.
2.3.3. Anti-Proliferative Activity
Anti-proliferative activity in the leaves, flowers, stems, and roots of S. palustris L. wastested in the A549 (lung adenocarcinoma), BxPC3 (pancreatic ductal adenocarcinoma), HT-29 and CACO-2 (colorectal adenocarcinoma), HCV29T (bladder cancer), and AML-NEV007(acute myeloid leukemia) cell lines (Figures 1 and 2). We chose tumors that are particularlyresistant to chemotherapy and difficult to treat. Cells were treated with ethanol extractfor 48 h, after which the MTS viability test and the analysis of the induction of apoptosiswere performed.
The most significant results were obtained for leaves and flower extracts of S. palustrisL. In particular, leaves extract markedly decreased the metabolic activity of all tested celllines to 8–22%. Extract from the flower showed weaker inhibition effects (10–52% remainingactivity). In addition, the roots and stems extracts were found weakest and reduced theviability to 30–90%. Diluted ethanol (1%) had no effect on cell lines.
We then tested the induction of apoptosis using Annexin V double staining and propid-ium iodide. As in the MTS test, the leaf and flower extracts significantly induced apoptosisin all tested cell lines in the range of 69–86%. The CACO-2 line was the most resistant,with the apoptosis level around 45%. A study by Kokhdan et al. [62] evaluated a methanolextract of S. pilifera against HT-29 cell line (colon adenocarcinoma) viability and demon-strated favorable inhibitory properties and significant anti-proliferative effects. It was alsoreported that S. laxa chloroform extract significantly prevented the proliferation of HT-29
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and T47D (ductal carcinoma) cell lines, and the total extract of S. subaphylla also showed anti-proliferative properties against T47D cell line [63]. Furthermore, Haznagy-Radnai et al. [7],noted that some Stachys species, such as S. palustris and S. recta, stems in methanolic ex-tracts displayed significant antiproliferative activity against cervix adenocarcinoma cells(HeLa); S. germanica flowers against breast adenocarcinoma (MCF-7) cells. In addition,Lachowicz et al. [11] noted that the methanolic extract of Sanguisorba officinalis L. leavesand flowers exhibited significant antiproliferative activity versus bladder cancer (HCV29T),colorectal adenocarcinoma (DLD-1), pancreatic ductal adenocarcinoma (BxPC3), and Jurkatcell lines. The obtained extracts contain a number of different substances that may have anadditive or synergistic anti-proliferative effect on cancer cells. Many of these substancesaffect various intracellular pathways that depend on the type of cancer cells [64]. Theexternalization of Annexin V indicates the activation of caspases. Most likely, the processof apoptosis follows the classical mitochondrial pathway. Polyphenolic compounds areabundant in the leaves and flowers of S. palustris L. Luteolin, ellagic acid, and apigeninderivatives are known for their activating effects on the p53 transcription factor. Theyalso affect the cell cycle and reduce the expression of the proapoptotic proteins from theBcl-2 family [65]. Polyphenols have anti-inflammatory properties by inhibiting the activityof COX2 and NFKB, which is important in cancer [66]. Some natural compounds haveexhibited synergism with established anticancer agents, and thus may reduce the sideeffects of chemotherapy. Further research is needed to identify active compounds and testtheir anti-cancer properties in appropriate in vivo models. The above results indicate thatthe anti-proliferative activity is mainly influenced by the part of the plant studied andmaterial extraction, as well as the profile of PCs.
Figure 1. The viability of different cancer cells after treatment with 30% ethanolic extracts for 48 h.One 1:10 solid-to-liquid (S/L) ratio was used for all parts of the plant. Obtained extracts were diluted30 times to final ethanol concentration 1%.
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Figure 2. Induction of apoptosis after treatment for 48 h.
2.4. Multivariate Analysis
The results show a significant relationship between the high value of particular groupsof the PCs and enzyme inhibitory activities, antioxidant, and antiproliferative activity,which was confirmed by the Pearson correlation coefficient. It is well known that PCsindicate numerous properties, including redox properties, and they, therefore, act as singletoxygen quenchers and hydrogen donors, as well as exhibiting antioxidant activity [50].Moreover, it is the synergistic effect between bioactive compounds in the tested materialthat provides antioxidant activity depending on, among others, their concentration [50].Thus, a very strong correlation was noted between the antioxidant potential (for ABTSand FRAP assay, respectively) and the overall value of PCs (R2 0.953 and 0.956), as wellas individual groups of substances, such as hydrolysable tannins (R2 0.967 and 0.901),polymeric procyanidins (R2 0.853 and 0.978), and phenolic acids (R2 0.792 and 0.685).
In addition, anthocyanins, flavan-3-ols, and flavonols had a stronger correlation withantiradical activity than FRAP assay (R2 0.699, 0.684, 0.699, respectively), but flavoneshad a stronger correlation with reducing power than ABTS assay (R2 0.700). A strongcorrelation between PCs and antioxidant assays was also reported by Bahadori et al. [41,60] and Khanaki et al. [45] in research concerning the analysis of Stachys cretica subsp.Mersinaea. On the other hand, the anthocyanins, flavan-3-ols, and flavonols showednegative correlation with BxPC3 cell line, and flavones noted negative correlation withα-glucosidase inhibitory activities.
Interestingly, PhGs, especially compounds identified in the roots, were the only groupof PCs to show a negative correlation with enzyme inhibitory activities, a negative strong
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correlation with antioxidant activity, regardless of the method used, and antiproliferativeactivity in the all cells line.
The strongest relationships to inhibition of α-amylase activity were noted for hydrolysabletannins, flavones, and PP against the inhibition of α-glucosidase activity—hydrolysable tannins,anthocyanins, flavan-3-ols, phenolic acid, and flavonols; whereas the strongest correla-tions in inhibition of pancreatic lipase activity were noted for phenolic acid and PP. Inturn, α-amylase inhibitory activities indicated a positive correlation with individual PCs,apart from p-coumaric acid and TPC (R2 −0.999) [41]. Furthermore, a strongly positivecorrelation was noted between the anti-proliferative potential (of all analyzed cancer cellsline) and hydrolysable tannins, flavones, PP, and TPC; while the remaining groups of thepolyphenolic compounds noted a positive correlation with cancer cells inhibitory activities,apart from PhGs.
Overall, the PCA results concerning different parts of plant extract data indicated aclear correlation with all PCs and anti-proliferative, anti-diabetic, anti-obesity, and antioxi-dant tests (Figure 3). The PCA detected two essential components that were responsiblefor 92.20% of data variance, including PC1 for 70.25%; while the second PC2 was onlyresponsible for 21.94%. Figure 3 clearly shows the tested parts of S. palustris L. based onphytochemicals and antioxidant, anti-proliferative, anti-obesity, and anti-diabetic activities:stems and roots are found on the left of the plot, flowers and leaves are distributed on theright part. The obtained data indicates that the metabolites that distinguish the studiedparts of the plant are the PCs and mainly hydrolyzed tannins and PP, which are foundin high amounts in the leaves and flowers (PC1), and PhGs, which are especially presentin the roots (PC2). Consequently, their type and concentration affect health-promotingproperties. Therefore, the biological activities demonstrated a difference between parts ofthe plant. Besides, the difference in the profile and concentration of PCs in plant fractionsis attributed to the difference in the morphological and anatomical structures and ongoingphysiological processes [44,67]. The results of the PCA method showed similar results asthe Pearson correlation.
Figure 3. PCA of bioactive compounds and biological activities for all parts of Stachys palustris L.
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3. Materials and Methods3.1. Chemicals, Material and Instruments
Chemicals: Acetonitrile UHPLC, methanol, ascorbic acid, formic acid, methanol, ABTS(2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid), 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), 2,4,6-tri(2-pyridyl)-s-triazine (TPTZ), 2,2-Di(4-tert-octylphenyl)-1-picrylhydrazyl (DPPH), methanol, acetic acid, α-amylase from porcine pancreas, α-glucosidasefrom Rhizopus sp., lipase from porcine pancreas, 3,5-dini-trosalicylic acid, Antibiotic-AntimycoticSolution, and RPMI 1640 culture medium were purchased from Sigma-Aldrich (Steinheim,Germany). (−)-Epicatechin, ellagic acid, dicaffeic acid, kampferol-3-O-galactoside, malvidin-3-O-glucoside, delphinidin 3-O-glucoside, cyanidin-3-O-glucoside, apigenin, apigenin 6-C-glucoside, and luteolin were purchased from Extrasynthese (Lyon, France). DMEM culturemedium with 10% FBS were purchased from Gibco (Thermo Fisher Scientific, Waltham, MA,USA), and MTS solution was purchased from Promega (Madison, WI, USA).
Material: Flowers, leaves, stems, and roots of Stachys palustris L. (~3 kg) were obtainedfrom a private garden in Szczytna (53◦33′46” N 20◦59′07” E), Lower Silesia, Poland. Theplant was collected randomly in August 2019 from different parts of the field (total area ofcultivation is 1 ha). The root of the Stachys palustris L. plant by Professor Ireneusz Ochmian.The fresh flowers, leaves, stems, and roots were directly frozen at −25 ◦C, and then freeze-dried and crashed. The powders were kept frozen (−25 ◦C) until planned analysis around2 weeks.
Color and shine of material were measured in a transmitted mode through Konica Mi-nolta CM-700d spectrophotometer in 1 cm-thick glass trays. Measurements were conductedin CIE L*a*b* system, through a 10◦ observer type and D65 illuminant [68].
3.3. Polyphenolic Compounds (PCs) by UPLC-ESI-TQD-MS/MS and Procyanidin Polymers (PP)by the Phloroglucinolysis Method
The method procedure was applied according to the protocol shared by Kapustaet al. [69]. Profiles of polyphenolic compounds were analyzed using UPLC-PDA-ESI-TQD-MS/MS. Briefly, the separation was carried out using a BEH C18 column (100 mm × 2.1 mmi.d., 1.7 µm, Waters, Warsaw, Poland) that was kept at 50 ◦C. For the anthocyanins investi-gation, the following solvent system was applied: mobile phase A (2% formic acid in water,v/v) and mobile phase B (2% formic acid in 40% ACN in water, v/v). For other polypheno-lic compounds, a lower concentration of formic acid was used (0.1%, v/v). The gradientprogram was set, as follows: 0 min 5% B, from 0 to 8 min linear to 100% B, and from 8 to9.5 min for washing and back to initial conditions. The injection volume of samples was5 µL (partial loop with needle overfill), and the flow rate was 0.35 mL/min. The followingparameters were used for TQD: capillary voltage 3.5 kV, cone voltage, 30 V in positive andnegative mode; the source was kept at 120 ◦C and the desolation temperature was 350 ◦C,con gas flow 100 L/h, and desolation gas flow 800 L/h. Argon was used as the collision gasat a flow rate of 0.3 mL/min. The profile compounds identification was based on specificPDA spectra, mass-to-charge ratio, and fragment ions obtained after collision-induceddissociation (CID). Quantification of compounds was achieved by the injection of solutionsof known concentrations that ranged from 0.05 to 5 mg/mL (R2 ≤ 0.9998) as standards((+)-catechin, 3,4-dicaffeoylquinic acid, luteolin 7-O-glucoside, apigenin 7-O-glucoside,
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kampferol-3-O-galactoside, ellagic acid, delphinidin 3-O-glucoside, cyanidin 3-O-glucoside,and malvidin 3-O-glucoside). All of the determinations were performed in duplicate andexpressed as mg/L. Waters MassLynx software v.4.1 was used for data acquisition and pro-cessing. The analysis of polyphenolic compounds were assayed in triplicate, and describedas mg per 100 g d.m.
The procyanidin polymers by the phloroglucinolysis method were applied accordingto the protocol shared by Lachowicz et al. [11]. The fractions of polymeric procyanidinwere analyzed using HPLC-FL. Briefly, the separation was carried out using a BEH C18RP column (2.1 × 5 mm, 1.7 µm; Waters Corporation, Milford, MA, USA) that was kept at15 ◦C with gradient elution of solvent A as 2.5% acetic acid and solvent B as acetonitrileat a flow rate of 0.42 mL/min for a duration of 10 min (100 mm × 2.1 mm i.d., 1.7 µm,Waters) that was kept at 30 ◦C, and the fluorescence was recorded at the excitation andemission wavelengths at 278 and 360 nm. Quantification of compounds was made fromprocyanidin B2, (+)-catechin and (−)-epicatechin (R2 ≤ 0.9995) as standards. The degree ofpolymerization was calculated as the molar ratio of all flavan-3-ol units (phloroglucinoladducts + terminal units) to (−)-epicatechin and (+)-catechin, which correspond to terminalunits. The analysis of polymeric procyanidins were assayed in triplicate, and described asmg per 100 g d.m.
3.4. In Vitro Biological Activity3.4.1. Extraction Procedure
The extraction was applied according to the protocol shared by Lachowicz et al. [11].Briefly, 0.2 g of dry material was mixed with 7 mL of 80% of methanol in water andsonicated at 20 ◦C for 20 min, and then incubated at 4 ◦C for 24 h, and the next step wascentrifuged. The supernatant was analyzed. The extractions method and all biologicalactivities were assayed in triplicate.
3.4.2. Antioxidant ActivityAntiradical Activity
The antiradical activity (ABTS) method was applied according to the protocol sharedby Re et al. [70]. Briefly, 0.03 mL of extracted material mixed with 2.97 mL of ABTS solutionand measured after 6 min at 734 nm using spectrophotometer. The result is described asmmol of Trolox equivalents (TE) per g d.m. (y = 33.64x + 2.68, R2 = 0.998).
Reducing Potency
The reducing potency (FRAP) method was applied according to the protocol sharedby Benzie and Strain [71]. Briefly, 0.1 mL of extracted material with 0.9 mL of distilledwater and 3 mL of ferric complex measured after 10 min of incubation at 593 nm usingspectrophotometer. The result is described as mmol of Trolox equivalents (TE) per g d.m(y = 19.82x − 1.85, R2 = 0.999).
3.4.3. Ability of α-Amylase, α-Glucosidase, Pancreatic Lipase Inhibitors
Ability of α-amylase, α-glucosidase inhibitors (anti-diabetic activity), and abilityof lipase activity inhibitors (anti-obesity activity) effect of the extracted material wereapplied according to the protocol shared by Nakai et al. [72], Podsedek et al. [73], andNickavar et al. [74]. Briefly, potato starch solution (0.2% (v/v)), the material extract, orphosphate buffer (0.1 M; pH 6.9; control) was mixed with α-amylase, and after incubation(37 ◦C, 10 min) the enzymatic reaction was stopped by the addition of HCl (0.4 M), theabsorbance was read at 600 nm. For the α-glucosidase assay, the material extract was mixedwith enzyme solution and incubated for 10 min. After that the reaction was initiated byp-nitrophenyl-α-D-glucopyranoside solution (5 mM), and incubated (37 ◦C, 20 min), andread the absorbance at 405 nm. For the pancreatic lipase assay, the material extracts weremixed with enzyme solution, and incubated (37 ◦C, 5 min). Then, methylumbelliferonesolution (0.1 mM) was added, and incubated (37 ◦C, 20 min) and read at an excitation
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wavelength of 360 nm and at an emission wavelength of 460 nm. The value of the inhibitor,required to inhibit 50% of the enzyme activity, was defined as the IC50 value. The IC50 ofthe fruits tested was obtained from the line of the plot of the fruit concentration in 1 mL ofreaction mixture versus the % inhibition.
3.4.4. Antiproliferative PotencyCell Lines and Cell Culture
The human cancer cell lines A549 (lung adenocarcinoma), BxPC3 (pancreatic ductaladenocarcinoma), HT-29, and CACO-2 (colorectal adenocarcinoma) and HCV29T (bladdercancer) were cultured in DMEM culture medium with 10% FBS (Gibco, Thermo FisherScientific, Walham, MA, USA) and Antibiotic Antimycotic Solution (Sigma-Aldrich, St.Louis, MO, USA). AML-NEV007 cell line (acute myeloid leukemia) was maintained inRPMI 1640 culture medium supplemented with 2 mM L-glutamine, 100 U/mL penicillinand 100 µg/mL streptomycin (Sigma-Aldrich, St. Louis, MO, USA), and 10% fetal bovineserum (FBS). All cell lines were cultured at 37 ◦C in a humidified atmosphere of 5% CO2.The cells were seeded at densities of 5 × 103 cells/0.1 mL (0.32 cm2) for cell viabilityassay. All cell lines were obtained from the collection of the Institute of Immunology andExperimental Therapy, Polish Academy of Sciences, Wroclaw, Poland.
Determination of Cell Viability
The plant extract was applied according to the protocol shared by Lachowicz et al. [11].Cell viability was assessed by the CellTiter 96 Aqueous One Solution Cell ProliferationAssay (Promega), according to the manufacturer’s protocol. Each treatment within a singleexperiment was performed in triplicate. Absorbance at 490 nm was recorded using a Wallac1420 VICTOR2 plate reader (PerkinElmer, Waltham, MA, USA). Data were normalized tothe untreated control.
Apoptosis Assay
Apoptosis was assessed by the Annexin V Apoptosis Detection Kit (Sigma-Aldrich,St. Louis, MO, USA), according to the manufacturer’s protocol. Briefly, the cells wereincubated with all compounds for 48 h, next were stained with Annexin V-FITC (8 µg/mL)and PI (5 µg/mL) for 15 min at RT in the dark. The cells were washed with cold PBS(with Ca2+ and Mg2+) containing 2.5% FBS between the steps. Data were acquired using aFACSCalibur flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA) and analyzedusing Flowing Software 2.5.1 (Perttu Terho, Turku, Finland). Apoptosis was quantified as apercentage of both Annexin V-positive and Annexin V/PI-double-positive cells.
3.5. Statistics
Statistical analysis included post-hoc Duncan’s multiple range test (p < 0.05) as one-way analysis. Principal component analysis (PCA) as the multivariate analysis wereperformed with Statistica 13.3 (StatSoft, Kraków, Poland).
4. Conclusions
Overall, the studies of PCs by the LC–PDA-ESI–TQD–MS/MS technique of differentparts of Stachys palustris L. detected 89 polyphenolic compounds, including 40 hydrolysabletannins, 16 phenylethanoid glycosides, 4 anthocyanins, 1 flavan-3-ol, 6 phenolic acids,1 flavonol, and 21 flavones. The profile and levels of these ingredients were conditional onthe parts of S. palustris L. used; thus, in flowers, roots, leaves, and stems, there were 56, 55,50, 49 compounds identified, respectively. The flavonols and anthocyanins were detectedonly in the flowers, while PhGs dominated the roots. In addition, the main compoundsevaluated in the research were vescalagin, cocciferin d2, isoverbascoside (verbascoside),luteolin 6-C-glucoside, luteolin 6-C-galactoside, apigenin 6-C-glucoside, (−)-epicatechin,ellagic acid, and malvidin 3-O-diglucoside.
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The highest amount of PCs was detected in the leaves, followed by the flowers, stems,and roots. The strongest antioxidant activity of the ABTS and FRAP assays for the leavesand flowers extracts were exhibited. In addition, the best digestive enzyme inhibitioneffect as a potential antidiabetic and anti-obesity activity. One of the most importantfeatures of leaf and flower extracts is their ability to induce cell death in various tumor celllines. In turn, in this study, the roots and stems were statistically the weakest in terms ofmedicinal potential.
For these reasons, S. palustris L. leaves and flowers rich in natural antioxidants withhigh biological activity should be further examined as health-beneficial ingredients forfunctional food, special food, cosmetics, and/or medical and pharmaceutical industries.Further investigations are required to isolate and identify active compounds from leavesand flowers with anti-microbiological and antiproliferative effects and a wider range ofantiproliferative effects, as well as the analysis of the bioavailability of compounds.
Supplementary Materials: The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/ph15070785/s1, Figure S1: LC-PDA-ESI-TQD-MS/MS chro-matogram fragile of the Stachys palustris L. roots extract at 280 and 320 nm; Table S1: Parameterof color.
Author Contributions: Conceptualization, S.L.-W. and A.P.-S.; Data curation, S.L.-W., A.R., I.K., T.C.,I.O. and A.P.-S.; Formal analysis, S.L.-W., A.R., I.K., T.C., I.O. and A.P.-S.; Funding acquisition, S.L.-W.,A.P.-S., P.R., W.Z.-S.; Investigation, S.L.-W., A.R., A.K., I.K., I.O. and A.P.-S.; Methodology, S.L.-W.,A.R., I.K., T.C. and I.O.; Software, S.L.-W., A.R., I.K., T.C. and I.O.; Supervision, S.L.-W. and A.P.-S.;Writing—original draft, S.L.-W., A.R. and A.P.-S. All authors have read and agreed to the publishedversion of the manuscript.
Funding: This research was partially supported by the National Science and Engineering ResearchCouncil of Canada (NSERC) Discovery grant (grant No. RGPIN-2018-04735).
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Acknowledgments: Authors acknowledge the financial support provided by the A. S. DekabanFoundation for supporting the stay of Sabina Lachowicz-Wisniewska as Visiting Assistant Professorat the UBC Food Process Engineering Laboratory, Vancouver, Canada (01.2020-05.2020). Supported bythe Foundation for Polish Science (FNP), and by the scholarship for young scientists of the Ministryof Education and Science (MEIN) for Sabina Lachowicz-Wisniewska.
Conflicts of Interest: The authors declare no conflict of interest.
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